Use of neurotoxin therapy for treatment of urologic and related disorders related to neurogenic bladder dysfunction

ABSTRACT

The present invention related to methods for treating neurological-urological conditions, including neurogenic bladder dysfunction. This is accomplished by administration of a botulinum toxin into the lower urinary tract of a patient with a neurogenic bladder dysfunction, including the bladder or urinary sphincter and the bladder wall.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/685,995, filed Oct. 14, 2003, which is a continuation ofU.S. patent application Ser. No. 09/978,982, filed Oct. 15, 2001, nowU.S. Pat. No. 6,667,041, which is a continuation of U.S. patentapplication Ser. No. 09/463,040, filed Jan. 17, 2000, now U.S. Pat. No.6,365,164, which is a 371 of PCT Application No. PCT/US98/14625, filedJul. 15, 1998, which claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application Ser. No. 60/052,580, filed Jul. 15, 1997. Theentire disclosure of each of the above-referenced applications isincorporated herein by reference.

FIELD OF THE INVENTION

The present invention provides methods for treating neuronally-mediatedurologic and related disorders, for example, neurogenic bladderdysfunction. This is accomplished by administering a composition thatincludes at least one neurotoxic compound such as botulinum toxin intothe lower urinary tract, including the bladder wall and the urinarysphincter or bladder neck, or by conventional therapies.

BACKGROUND OF THE INVENTION

Many medical conditions in urology are rooted in a spastic dysfunctionof the sacral reflex arcs. Examples of such conditions include pelvicpain (e.g., interstitial cystitis, endometriosis, prostatodynia,urethral instability syndromes), pelvic myofascial elements (e.g.,levator sphincter, dysmenorrhea, anal fistula, hemorrhoid), urinaryincontinence (e.g., unstable bladder, unstable sphincter), prostate(disorders (e.g., BPH, prostatitis, prostate cancer), recurrentinfection (secondary to spastic sphincter, hypertrophied bladder neck)and neurogenic bladder dysfunction (e.g., Parkinson's Disease, spinalcord injury, stroke, multiple sclerosis, spasm reflex).

Neurogenic bladder dysfunction is a dysfunction that results frominterference with the normal nerve pathways associated with urination.Normal bladder function is dependent on the nerves that sense thefullness of the bladder and on those that trigger the muscle movementsthat either empty it or retain urine. Normally, the reflex to urinate istriggered when the bladder fills to 300-500 ml. The bladder is thenemptied when the contraction of the bladder wall forces urine outthrough the urethra. The bladder, internal sphincters, and externalsphincters may all be affected by nerve disorders that createabnormalities in bladder function. The damage can cause the bladder tobe underactive, in which it is unable to contract and unable to emptycompletely, or it can be overactive, in which it contracts too quicklyor frequently.

One type of neurogenic bladder dysfunction is overactive (spastic orhyper-reflexive) bladder. An overactive neurogenic bladder ischaracterized by uncontrolled, frequent expulsion of urine from thebladder. There may be reduced bladder capacity and incomplete emptyingof urine. Spastic bladder may be caused by an inability of the detrusormuscle of the bladder to inhibit emptying contractions until areasonable amount of urine has accumulated. Often, a strong urge to voidis experienced when only a small amount of urine is in the bladder. Thepatient may report symptoms of urgency, frequency, nocturia, andincontinence. Another type of neurogenic bladder dysfunction ischaracterized by difficulty in relaxing the urinary sphincter muscle.The sphincter may be spastic. This causes difficulty in emptying thebladder, which can lead to urinary retention and urinary tractinfections. In another type of neurogenic bladder dysfunction, both thedetrusor muscle and the urinary sphincter simultanously contractresulting in urinary retention. A dysfunction associated withsimultaneous contraction of both the detrusor and the urinary sphincteris called detrusor-external sphincter dyssynergia (DESD).

There are numerous causes for neurogenic bladder dysfunction. Anoveractive bladder/sphincter may be caused by interruptions in the nervepathways to the bladder occurring above the sacrum. This nerve damageresults in a loss of sensation and motor control and is often seen instroke, Parkinson's disease, spina bifida, diabetes, pelvic surgery, orinvertebral disc herniation, and most forms of spinal cord injuries.Sometimes no cause is found, and such idiopathic cases may be due toanxiety or aging.

Inability to control urination, also known as urinary incontinence, isperhaps the most common symptom associated with the neurogenic bladder.This may be caused by abnormalities in bladder capacity or malfunctionof control mechanisms such as the bladder neck and/or external urethralsphincter muscle that are important for the bladder's storage function.However, not all neurogenic bladder dysfunctions are characterized byurinary incontinence.

Symptoms including a dribbling urinary stream, straining duringurination or inability to urinate may also be associated with neurogenicbladder. Urinary retention may result either from loss of bladder musclecontracting performance or loss of appropriate coordination between thebladder muscle and the external urethral sphincter muscle.

Irritating symptoms, such as urinary frequency and urgency, may beevidence of bladder hyperactivity. Other irritating symptoms may includepainful urination (dysuria), which may be a result of a urinary tractinfection (UTI) caused by urine being held too long in the bladder. UTIwith fever is a sign of potential severe kidney infection(pyelonephritis) and is a more worrisome situation as it may result inpermanent damage of the kidney(s).

Stones may also form in the urinary tract of individuals with aneurogenic bladder dysfunction caused by the stoppage of urine flowand/or infection.

Abnormal backup of urine from the bladder to the kidney(s), also knownas vesicoureteral reflux (VUR), may develop as a means of releasing highpressure within the bladder. A UTI is of particular concern as VUR mayplace the patient at significant risk for a severe kidney infection bytransporting infected bladder urine directly to the kidney(s).

In the past, physicians have tried a number of treatments for neurogenicbladder dysfunction. One treatment option is to use drugs to relax thebladder, such as anticholinergics such as propantheline and oxybutynin.However, these drugs do not act specifically towards the bladder andtend to reduce muscle tone throughout the body, an undesirable sideeffect. Intermittent catheterization is another treatment that can beused to empty the bladder; however, it often requires a skilledcaregiver to place the catheter and is inconvenient to the patient. Anindwelling catheter is another option; however, such catheters haveseveral risks including infections and bladder stones. Another treatmentoption is to simply have the patient wear diapers or other protectivetype devices to prevent urine leakage and overflow from wetting clothingand/or bedding.

Another embodiment of the present invention relates to another type ofurological-neurological dysfunction associated with the prostate. Theprostate is a partially glandular and partially fibromuscular organ ofthe male reproductive system. During aging, the prostate tends toenlarge (hypertrophy). This prostatic enlargement can lead to urethralobstruction and voiding dysfunction.

Prostatic enlargement is a common occurrence in older men. Lytton et al.(Lytton, B., Emery, J. M and Harvard, B. M. [1973] 99: 639-645)estimated that a 45 year old male had a 10% risk of prostate surgery byage 70. The U.S. Census Report estimates that there are 30 millionpeople today over age 65. This segment of the population is projected torise to 50 million over the next 30 years. Therefore, the number of menwith prostatic enlargement also will increase. According to draftreports of the National Kidney and Urologic Disease Advisory Board,425,000 prostatectomies were performed in the United States in 1989.Based on population growth estimates, the number of prostatectomiesperformed annually will rise to 800,000/year by the year 2020.

The urethra passes through the prostate (prostatic urethra) as itcourses to the external urethral orifice. The prostate has five distinctlobes that are apparent at 12 weeks in the human fetus (Lowsley, O. S.Am. J. Anat. [1912] 13: 299-349.). Although the lobular branching foundin the fetus is not visible in the prepubescent prostate, the lateralmiddle anterior and posterior lobes are used to describe the enlargedprostate.

A more recent viewpoint is that the prostate also is comprised ofseveral morphologically distinct zones (McNeaL, J. Urol. Clin. North Am.[1990] 17(3): 477-486). The majority of the glandular volume is composedof the peripheral zone (˜70-75%). The remainder of glandular volume isdivided into the central zone (˜20-25%), the transition zone (˜5-10%)and the periurethral glandular zone (˜1%).

McNeal (1990) reported that BPH develops in the transition zone and theperiurethral glandular zone. BPH nodules develop either within orimmediately adjacent to the preprostatic sphincteric zone. Thetransition zone is a small region close to the urethra intimatelyrelated to the proximal urethral sphincter. The stroma of the transitionzone is dense and compact, and is unusually susceptible toneurologically-induced disturbances of growth control. Its glandspenetrate the sphincter, while sphincter muscle fibers penetrate thetransition stroma. The periurethral glandular zone has a similarurogenic sinus origin as the transition zone.

BPH may be associated with increased amounts of stroma relative toepithelium (Bartsch, G., Muller, H. R., Oberholzer, M, Rohr, H., P., J.Urol. [1979] 122: 487-491). A significant portion of the stroma issmooth muscle (McNeal, 1990) which is under sympathetic nervous control.The contractile properties of this smooth muscle could account for thedynamic component of obstruction in BPH (Bruschini, H. et at. [1978]Invest. Urol. 15(4): 288-90; Lepor, H [1990] Urol. Clin. North Am.17(3): 651-658).

In addition to sympathetic control of prostatic stroma, the prostate ishighly innervated. The prostate nerve fibers enter the prostate from theposterior lateral aspect, with a concentration of ganglia near thejunction between the prostate and the seminal vesicles (Maggi, C. A, ed.[1993] Nervous control of the Urogenital System, Harwood AcademicPublishers; Higgins, J. R. A. and Gosling, J. A. [1989] Prostate Suppl.2: 5-16).

Acetylcholine (ACH), neuropeptide Y (NPY), vasoactive intestinal peptide(VIP) and noradrenaline fibers have been described in this gland. A richplexus of ACH-positive nerve cell bodies is associated with secretoryacini in all parts of the gland. Some of the ACH fibers also contain NPYneurons. VIP-containing neurons have been found associated withACH-containing nerve cell bodies. Occasional neurons have been foundbetween the ACH-staining nerve fibers, suggesting that both NPY andnoradrenergic neurons supply smooth muscle (Higgins, J. R. A andGosling, J. A [1989] Prostate Suppl. 2: 5-16).

Autonomic nerves are distributed evenly between the central andperipheral zones of the prostate (Higgins, J. R. A. and Gosling, J. A[1989] Prostate Suppl. 2: 5-16). Peripheral neuronal control is similar.In addition, there is no difference in the type of nerve fibers, foundassociated with either epithelial or stromal elements of the gland.

The anatomical studies of nerve fiber types in the prostate, coupledwith other studies of innervation of prostatic stroma (Brushing H,Schmidt, R. A, Tanagho, E. A, [1978] Invest. Urol. 15(4): 288-290;Watanabe, H., Shima, M., Kojima, M., Ohe, H. L. [1989] Pharmacol. Res.21 (Suppl. 2): 85-94) suggest that cholinergic innervation influencesepithelial behavior, while adrenergic innervation influences stromaltonus (excitability). These observations have provided a rationale forthe use of, for example, alpha blockers in the treatment of BPH. Theeffects of alpha blockers (Downie, J. W. and Bialik, G. J. [1988] J.Pharmacal. Exp. Ther. 246(1): 352-358) can also account for improvementsin symptoms of BPH as a result of dampening of dysfunctional striatedsphincter behavior by the alpha blockers.

Studies have also shown that there are several tachykinins (for example,substance P [SP], calcitonin gene related peptide [CGRP], neurokinin A,bradykinin, and nerve growth factor [NGF]) that can influence the tonusof smooth muscle (Hakanson, et al., [1987] Neuroscience 21(3): 943-950).Neurotransmitter receptors have been quantified throughout the prostate(e.g., NPY, VIP, SP, leu-enkephalin (L-enk), met-enkephalin, 5-HT,somatostatin, acetylcholinesterase positive fibers (ACTH), and dopaminebeta-hydroxylase (DBH) (Crowe, R., Chapple, C. R., Burnstock, G. TheHuman Prostate Gland: A Histochemical and Immunohistochemical Study ofNeuropeptides, Serotonins, Dopamine beta-Hydroxylase andAcetylcholinesterase in Autonomic Nerves and Ganglia). There is somevariation in receptor density at different prostatic sites in benignprostatic hyperplasia.

Changes in electrophysiologically recorded cellular behavior and inconcentration of neuropeptides within the spinal cord have been shown tobe a secondary consequence of mechanical pinch to the tail muscles of arat, catheter stimulation of the posterior urethra, andelectrostimulation of a peripheral nerve. Dyssynergia between thedetrusor and the urethral sphincter is a significant finding inprostatodynia patients. Denervation of the prostate has been shown toproduce dramatic changes within the prostatic epithelium. Thus there isevidence that experimentally induced alterations in neurologicalinfluences can be produced in the sacral, spinal cord, bladder orurethra through mechano-, electro-, chemical or thermal (microwave,laser) methods to change irritative behavior. However, there have beenno known attempts to use neurotoxins for therapeutic applications.

There is poor correlation between the degree of prostatic enlargementand the severity of symptoms. While 80% of men age 70 show BPH ontransrectal ultrasound scans, only 20% seek surgery (Coffey, D. S. andWalsh, P. C. [1990] Urol. Clin. North Am. 17(3): 461-475), the treatmentof choice for BPH (Fowler, F. J. Jr., Wennberg. J. E., Timothy, R. P.[1988] J. Amer. Med. Assoc. 259(20): 3022-3028). Symptoms of irritationmay far exceed symptoms expected based on the size of the prostate.Symptoms may improve after surgical treatment of BPH by procedures suchas transurethral resection of the prostate (TURF) (Christensen, Aagaard,M. M. J., Madsen, P.O. [1990] Urol. Clin. North Am. 17(3): 621-629),balloon dilation (Dowd, J. B. and Smith, J. J. III [1990] Urol. Clin.North Am. 17(3): 671-677), or prostatic hyperthermia (Baert, L., Ameye,F., Willemen, P., et al. [1990] J. Urol. 144: 1383-1386). However,symptoms persist in as many as 15% of all BPH patients (Baert, L.,Ameye. F., Willemen, P., et al. [1990] J. Urol. 144: 1383-1386;Wennberg, J. E., Mully, A. G., Hanley, D., Timothy, R. P., Fowler, F.J., Roos, R. P., Barry, M. J. et al. [1988] J. Amer. Med. Assoc. 259:3027-3030). Up to 25% of BPH patients have secondary procedures in longterm follow-up studies, suggesting that surgical approaches do notaddress the fundamental mechanisms that produce BPH, i.e., the faultyneurological influence (control mechanism) on the integrity of the lowerurinary tract.

The need for repeated surgeries, the morbidity and mortality associatedwith TURP and the cost of surgery have led to the development of somenon-surgical approaches such as androgen ablation (McConnell. J. D.,[1990] Urol. Clin. North Am. 11(3): 661-670) and the use of alphablockers discussed above, but few medical or surgical treatments to datehave produced a restoration of void behavior to normal state (flow rateof about 25 cc/sec and void volume of about 400 cc).

OBJECTS AND SUMMARY OF THE PRESENT INVENTION

The present invention uses chemical and non-chemical methods,particularly neurotoxins, to modulate neuronally-mediated urologic andrelated disorders, such as neurogenic bladder dysfunction. Such methodscan be also be used to treat BPH and related conditions such asprostatitis. The instant invention also may remove triggers of changesin the CNS; by non-chemical methods including biofeedback, or bychemical methods that treat BPH and other urological conditions by theadministration of substances that block various neurological activities,such as, for example, selected neurotoxins including botulinum toxin.

It is an object of the instant invention to provide safe, inexpensive,out-patient methods for the prevention and treatment ofurological-neurological dysfunctional states or conditions, for example,prostatic enlargement.

It is a further object of the present invention to provide compositionsfor this therapeutic goal. It is a still further object of the presentinvention to provide dosages and methods of administration forcompositions useful for the prevention and treatment ofneurological-urological conditions.

In accordance with one aspect of the present invention, there areprovided methods of treating urological-neurological conditions,including neurogenic bladder dysfunction, in mammals, said methodscomprising the step of administering a therapeutically effective amountof at least one neurotoxin to such a mammal. It is preferred that theneurotoxin inhibits synaptic function. Such inhibition producesselective denervation, and, for example, atrophy of the prostate andreversal of irritative symptoms associated with prostatic enlargement.In one embodiment of the instant invention, the neurotoxin inducesdysfunction of the presynaptic neuronal terminal by specific binding andblockade of acetylcholine release at myoneural junctions and atcholinergic junctions generally. Such a neurotoxin can be, for example,botulinum toxin type A (BOTOX®, Allergan).

In accordance with another aspect of the present invention, a method isprovided for treating a specific urological-neurological condition.Preferably, the urological-neurological condition is neurogenic bladderdysfunction. In one aspect, the neurogenic bladder dysfunction is notcharacterized by the symptom of urinary retention. This method comprisesthe step of administering a therapeutic amount of a botulinum toxin intothe urinary sphincter of a patient with neurogenic bladder dysfunction,thereby relieving a symptom of said neurogenic bladder dysfunction. In apreferred embodiment, a symptom of neurogenic bladder dysfunctionincludes recurrent bladder infection, incontinence, and urgeincontinence. The neurogenic bladder dysfunction can include a spasticsphincter.

In another embodiment of the present invention, the method for treatinga neurogenic bladder dysfunction comprises the step of administering atherapeutic amount of a botulinum toxin into the bladder wall of apatient with neurogenic bladder dysfunction, thereby relieving a symptomof said neurogenic bladder dysfunction. In a preferred embodiment, asymptom of neurogenic bladder dysfunction includes urinary retention,recurrent bladder infection, incontinence, and urge incontinence. Theneurogenic bladder dysfunction can include a spastic bladder. Theneurogenic bladder dysfunction may be secondary to a disease conditionselected from the group consisting of Parkinson's disease, a spinal cordinjury, a stroke, multiple sclerosis, and a spasm reflex. Preferably,treatment according to methods of the present invention result inincreased bladder capacity.

Preferably, the neurotoxin is safe, highly selective and easy todeliver, including when combined with other therapies. Other usefulneurotoxins include capsaicin, resinoferatoxin and a-bungotoxin.Delivery of the neurotoxin can be by any suitable means. A convenientand localized method of delivery is by injection.

A therapeutically effective amount of the neurotoxin is the dosagesufficient to inhibit neuronal activity for at least one week, morepreferably one month, most preferably for approximately 6 to 8 months orlonger. Dosing can be single dosage or cumulative (serial dosing), andcan be readily determined by one skilled in the art. Neurotoxin can bedelivered serially (i.e., one time per month, one time per every sixmonths) so that the therapeutic effect can be optimized. Such a dosageschedule is readily determined by one skilled in the art based on, e.g.,patient size and the condition to be treated, and will depend on manyfactors, including the neurotoxin selected, the condition to be treated,the degree of irritation, and other variables. One suggested course oftreatment for BPH is 200 units of BOTOX® every 3-4 months, as indicatedby therapeutic requirements. Other suggested dosages of botulinum toxinsare discussed in detail below.

The aforementioned methods of treatment should be particularly usefulfor the long-term control of neurological-urological disorders, such asthe symptoms of neurogenic bladder dysfunction and the symptoms ofprostatic enlargement, without the need for surgical intervention.Furthermore, the methods of the instant invention provide for control ofneurological-urological disorders, e.g., BPH and related conditions, ina highly selective manner, without the potential side effects andtreatment failures associated with current treatment modalities.

Other objects of the present invention will be readily apparent to thoseof ordinary skill in the art.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

“Urological-neurological condition or disorder” includes many medicalconditions in urology rooted in a spastic dysfunction and/ordegeneration of the sacral reflex arcs. Examples of such conditionsinclude pelvic pain (e.g., interstitial cystitis, endometriosis,prostatodynia, urethral instability syndromes), pelvic myofascialelements (e.g., levator sphincter, dysmenorrhea, anal fistula,hemorrhoid), urinary incontinence (e.g., motor or sensory, unstablebladder, unstable sphincter), prostate disorders (e.g., BPH, prostatecancer), recurrent infection (secondary to sphincter spasticity), andurinary retention (secondary to spastic sphincter, hypertrophied bladderneck), and neurogenic bladder dysfunction (e.g., Parkinson's Disease,spinal cord injury, stroke, multiple sclerosis, spasm reflex) and othersuch urological conditions of a nervous etiology.

The prostatic enlargement that can be treated according to the methodsof the instant invention can be of any etiology. The instant inventionis particularly suited for the treatment of prostatic hyperplasia,especially benign prostatic hyperplasia. The present invention can alsobe used for the treatment of enlargement of the prostate withinflammation (prostatitis), particularly abacterial prostatitis. Inaddition, the methods of the instant invention can be used for thetreatment of prostatodynia.

In accordance with another aspect of the present invention, a method isprovided for treating a urological-neurological condition. Preferably,the urological-neurological condition is neurogenic bladder dysfunction.In one embodiment, preferred neurogenic bladder dysfunctions to treatinclude dysfunctions where only the bladder (detrusor muscle) isspastic, i.e., there is no sphincter involvement; and/or dysfunctionswhere only the sphincter is spastic, i.e., there is no bladder (detrusormuscle) involvement. In another embodiment, preferred neurogenic bladderdysfunctions to treat include dysfunctions where there is a combineddysfunction, i.e., both the bladder and the urinary sphincter(s) spasmor contract simultaneously.

In one aspect of this embodiment, the method comprises the step ofadministering a therapeutic amount of a botulinum toxin into the urinarysphincter of a patient with neurogenic bladder dysfunction, therebyrelieving a symptom of said neurogenic bladder dysfunction. Anotherpreferred symptom of a neurogenic bladder dysfunction to relieve isincreased incidents of UTIs (urinary tract infections) or recurrentinfection. Another preferred symptom to relieve includes urgeincontinence and incontinence. Methods of the present invention mayrelieve (i.e. treat, reduce, eliminate, or minimize) the symptoms ofneurogenic bladder dysfunction. Preferably, a neurogenic bladderdysfunction to treat to treat by the method of administering into aurinary sphincter is primarily characterized by urinary incontinence. Inone embodiment, a preferred neurogenic bladder dysfunction to treat bythe method of administering into a urinary sphincter does not includeneurogenic bladder dysfunctions characterized by urinary retention.

Methods of the present invention are useful for easing the difficultysome neurogenic bladder dysfunction patients have in relaxing theurinary sphincter muscle. Difficulty in relaxing the urinary sphinctermuscle causes difficulty in emptying the bladder, which can lead tourinary retention and recurrent urinary tract infections. Causes ofurinary sphincter spasm include interruptions in the nerve pathways tothe bladder occurring above the sacrum. This nerve damage results in aloss of sensation and/or motor control and is often seen in stroke,Parkinson's disease, spina bifida, diabetes, pelvic surgery, orinvertebral disc herniation, and most forms of spinal cord injuries.Causes for the neurogenic dysfunction may be unknown, thus neurogenicdysfunction which may be treated by the present invention may alsoinclude idiopathic cases, including those due to anxiety or aging.

In another aspect of this embodiment of the present invention, themethod for treating a neurogenic bladder dysfunction comprises the stepof administering a therapeutic amount of a botulinum toxin into thebladder wall of a patient with a neurogenic bladder dysfunction, therebyrelieving a symptom of the neurogenic bladder dysfunction. A neurogenicbladder dysfunction which is treatable by methods of the inventionincludes bladder spasticity. Preferred symptoms to relieve using theinstant methods include urge-type incontinence, recurrent bladderinfection, incontinence, and urinary retention. Causes of neurogenicspastic bladder and urge-type dysfunction include interruptions in thenerve pathways to the bladder occurring above the sacrum. This nervedamage can result in a loss of sensation and/or motor control and isoften seen in stroke, Parkinson's disease, spina bifida, diabetes,pelvic surgery, or invertebral disc herniation, and most forms of spinalcord injuries. Causes for the neurogenic dysfunction may be unknown,thus neurogenic dysfunction which may be treated by the presentinvention may also include idiopathic cases, including those due toanxiety or aging. In a preferred embodiment, bladder capacity isincreased by treatment according to the methods of the presentinvention.

The methods of the present invention preferably comprise theadministration of a botulinum toxin. The botulinum toxin useful formethods of the present invention include botulinum toxin type A,botulinum toxin type B, botulinum toxin type C, botulinum toxin type D,botulinum toxin type E, botulinum toxin type F, and botulinum toxin typeG. Preferred botulinum toxins to use includes botulinum toxin type A andbotulinum toxin type B. Preferably, botulinum toxin type A is used, andeven more preferably, BOTOX®, is used. Other forms of botulinum toxinthat are compatible with the present invention include chimeric orhybrid botulinum toxins, see, e.g., U.S. Pat. No. 5,939,070, which isincorporated by reference herein in its entirety; recombinantly madebotulinum toxins, see, e.g., U.S. Pat. No. 5,919,665, which isincorporated by reference herein in its entirety; and retargetedbotulinum toxins, see, e.g., U.S. Pat. Nos. 5,989,545 and/or 6,461,617,which are incorporated by reference herein in their entirety. Retargetedbotulinum toxins refer to botulinum toxins that are attached to anon-native targeting moiety with affinity for the selected targettissue. Formulations of botulinum toxin that are compatible with thepresent invention include depot formulations, e.g., polymeric implants,microspheres, wafers, and gels, for sustained or controlled release.See, e.g., U.S. Pat. Nos. 6,585,993; 6,506,399; 6,306,423; 6,312,708;6,383,509, all of which are incorporated herein by reference in theirentirety.

The genus Clostridium has more than one hundred and twenty sevenspecies, grouped according to their morphology and functions. Theanaerobic, gram positive bacterium Clostridium botulinum produces apotent polypeptide neurotoxin, botulinum toxin, which causes aneuroparalytic illness in humans and animals referred to as botulism.The spores of Clostridium botulinum are found in soil and can grow inimproperly sterilized and sealed food containers of home basedcanneries, which are the cause of many of the cases of botulism. Theeffects of botulism typically appear 18 to 36 hours after eating thefoodstuffs infected with a Clostridium botulinum culture or spores. Thebotulinum toxin can apparently pass unattenuated through the lining ofthe gut and attack peripheral motor neurons. Symptoms of botulinum toxinintoxication can progress from difficulty walking, swallowing, andspeaking to paralysis of the respiratory muscles and death.

Botulinum toxin type A is the most lethal natural biological agent knownto man. About 50 picograms of a commercially available botulinum toxintype A (purified neurotoxin complex) is a LD₅₀ in mice (i.e. 1 unit).One unit of BOTOX® contains about 50 picograms (about 56 attomoles) ofbotulinum toxin type A complex. Interestingly, on a molar basis,botulinum toxin type A is about 1.8 billion times more lethal thandiphtheria, about 600 million times more lethal than sodium cyanide,about 30 million times more lethal than cobra toxin and about 12 milliontimes more lethal than cholera. Singh, Critical Aspects of BacterialProtein Toxins, pages 63-84 (chapter 4) of Natural Toxins II, edited byB. R. Singh et al., Plenum Press, New York (1976) (where the stated LD₅₀of botulinum toxin type A of 0.3 ng equals 1 U is corrected for the factthat about 0.05 ng of BOTOX® equals 1 unit). One unit (U) of botulinumtoxin is defined as the LD₅₀ upon intraperitoneal injection into femaleSwiss Webster mice weighing 18 to 20 grams each.

Seven generally immunologically distinct botulinum neurotoxins have beencharacterized, these being respectively botulinum neurotoxin serotypesA, B, C₁, D, E, F and G, each of which is distinguished byneutralization with type-specific antibodies. The different serotypes ofbotulinum toxin vary in the animal species that they affect and in theseverity and duration of the paralysis they evoke. For example, it hasbeen determined that botulinum toxin type A is 500 times more potent, asmeasured by the rate of paralysis produced in the rat, than is botulinumtoxin type B. Additionally, botulinum toxin type B has been determinedto be non-toxic in primates at a dose of 480 U/kg which is about 12times the primate LD₅₀ for botulinum toxin type A. Moyer E et al.,Botulinum Toxin Type B: Experimental and Clinical Experience, beingchapter 6, pages 71-85 of “Therapy With Botulinum Toxin”, edited byJankovic, J. et al. (1994), Marcel Dekker, Inc. Botulinum toxinapparently binds with high affinity to cholinergic motor neurons, istranslocated into the neuron and blocks the release of acetylcholine.Additional uptake appears to take place through lower affinityreceptors, as well as by the process of phagocytosis and or pinocytoscisin many types of cells.

Regardless of serotype, the molecular mechanism of toxin intoxicationappears to be similar and to involve at least three steps or stages. Inthe first step of the process, the toxin binds to the presynapticmembrane of the target neuron through a specific interaction between theheavy chain, H chain, and a cell surface receptor; the receptor isthought to be different for each type of botulinum toxin and for tetanustoxin. The carboxyl end segment of the H chain, H_(C), appears to beimportant for targeting of the toxin to the cell surface.

In the second step, the toxin crosses the plasma membrane of thepoisoned cell. The toxin is first engulfed by the cell throughreceptor-mediated endocytosis, and an endosome containing the toxin isformed. The toxin then escapes the endosome into the cytoplasm of thecell. This step is thought to be mediated by the amino end segment ofthe H chain, H_(N), which triggers a conformational change of the toxinin response to a pH of about 5.5 or lower. Endosomes are known topossess a proton pump which decreases intra-endosomal pH. Theconformational shift exposes hydrophobic residues in the toxin, whichpermits the toxin to embed itself in the endosomal membrane. The toxin(or at a minimum the light chain) then translocates through theendosomal membrane into the cytoplasm.

The last step of the mechanism of botulinum toxin activity appears toinvolve reduction of the disulfide bond joining the heavy chain, Hchain, and the light chain, L chain. The entire toxic activity ofbotulinum and tetanus toxins is contained in the L chain of theholotoxin; the L chain is a zinc (Zn++) endopeptidase which selectivelycleaves proteins essential for recognition and docking ofneurotransmitter-containing vesicles with the cytoplasmic surface of theplasma membrane, and fusion of the vesicles with the plasma membrane.Tetanus neurotoxin, botulinum toxin types B, D, F, and G causedegradation of synaptobrevin (also called vesicle-associated membraneprotein (VAMP)), a synaptosomal membrane protein. Most of the VAMPpresent at the cytoplasmic surface of the synaptic vesicle is removed asa result of any one of these cleavage events. Botulinum toxin serotype Aand E cleave SNAP-25. Botulinum toxin serotype C1 was originally thoughtto cleave syntaxin, but was found to cleave syntaxin and SNAP-25. Eachof the botulinum toxins specifically cleaves a different bond, exceptbotulinum toxin type B (and tetanus toxin) which cleave the same bond.Each of these cleavages block the process of a vesicle-merging with themembrane docking, thereby preventing of the cell so that the contents ofthe vesicle will be ejected from the cell. This process is calledexocytosis of vesicle content.

Botulinum toxins have been used in clinical settings for the treatmentof neuromuscular disorders characterized by hyperactive skeletal muscles(i.e. motor disorders). In 1989 a botulinum toxin type A complex hasbeen approved by the U.S. Food and Drug Administration for the treatmentof blepharospasm, strabismus and hemifacial spasm. Subsequently, abotulinum toxin type A was also approved by the FDA for the treatment ofcervical dystonia and for the treatment of glabellar lines, and abotulinum toxin type B was approved for the treatment of cervicaldystonia. Non-type A botulinum toxin serotypes apparently have a lowerpotency and/or a shorter duration of activity as compared to botulinumtoxin type A. Clinical effects of peripheral intramuscular botulinumtoxin type A are usually seen within one week of injection. The typicalduration of symptomatic relief from a single intramuscular injection ofbotulinum toxin type A averages about three months, althoughsignificantly longer periods of therapeutic activity have been reported.

Although all the botulinum toxins serotypes apparently inhibit releaseof the neurotransmitter acetylcholine at the neuromuscular junction,they do so by affecting different neurosecretory proteins and/orcleaving these proteins at different sites. For example, botulinum typesA and E both cleave the 25 kiloDalton (kD) synaptosomal associatedprotein (SNAP-25), but they target different amino acid sequences withinthis protein. Botulinum toxin types B, D, F and G act onvesicle-associated protein (VAMP, also called synaptobrevin), with eachserotype cleaving the protein at a different site. Finally, botulinumtoxin type C₁ has been shown to cleave both syntaxin and SNAP-25. Thesedifferences in mechanism of action may affect the relative potencyand/or duration of action of the various botulinum toxin serotypes.Apparently, a substrate for a botulinum toxin can be found in a varietyof different cell types. See e.g. Biochem, J 1;339 (pt 1):159-65:1999,and Mov Disord, 10(3):376:1995 (pancreatic islet B cells contains atleast SNAP-25 and synaptobrevin).

The molecular weight of the botulinum toxin protein molecule, for allseven of the known botulinum toxin serotypes, is about 150 kD. In vitrostudies have indicated that botulinum toxin inhibits potassium cationinduced release of both acetylcholine and norepinephrine from primarycell cultures of brainstem tissue. Additionally, it has been reportedthat botulinum toxin inhibits the evoked release of both glycine andglutamate in primary cultures of spinal cord neurons and that in brainsynaptosome preparations botulinum toxin inhibits the release of each ofthe neurotransmitters acetylcholine, dopamine, norepinephrine (HabermannE., et al., Tetanus Toxin and Botulinum A and C Neurotoxins InhibitNoradrenaline Release From Cultured Mouse Brain, J Neurochem51(2);522-527:1988) CGRP, substance P and glutamate (Sanchez-Prieto, J.,et al., Botulinum Toxin A Blocks Glutamate Exocytosis From Guinea PigCerebral Cortical Synaptosomes, Eur J. Biochem 165;675-681:1987. Thus,when adequate concentrations are used, stimulus-evoked release of mostneurotransmitters is blocked by botulinum toxin. See e.g. Pearce, L. B.,Pharmacologic Characterization of Botulinum Toxin For Basic Science andMedicine, Toxicon 35(9);1373-1412 at 1393; Bigalke H., et al., BotulinumA Neurotoxin Inhibits Non-Cholinergic Synaptic Transmission in MouseSpinal Cord Neurons in Culture, Brain Research 360;318-324:1985;Habermann E., Inhibition by Tetanus and Botulinum A Toxin Of the releaseof [ ³ H]Noradrenaline and [ ³ H]GABA From Rat Brain Homogenate,Experientia 44;224-226:1988, Bigalke H., et al., Tetanus Toxin andBotulinum A Toxin Inhibit Release and Uptake of Various Transmitters, asStudied with Particulate Preparations From Rat Brain and Spinal Cord,Naunyn-Schmiedeberg's Arch Pharmacol 316;244-251:1981, and; Jankovic J.et al., Therapy With Botulinum Toxin, Marcel Dekker, Inc., (1994), page5.

Botulinum toxin type A can be obtained by establishing and growingcultures of Clostridium botulinum in a fermenter and then harvesting andpurifying the fermented mixture in accordance with known procedures. Allthe botulinum toxin serotypes are initially synthesized as inactivesingle chain proteins which must be cleaved or nicked by proteases tobecome neuroactive. The bacterial strains that make botulinum toxinserotypes A and G possess endogenous proteases and serotypes A and G cantherefore be recovered from bacterial cultures in predominantly theiractive form. In contrast, botulinum toxin serotypes C₁, D and E aresynthesized by nonproteolytic strains and are therefore typicallyunactivated when recovered from culture. Serotypes B and F are producedby both proteolytic and nonproteolytic strains and therefore can berecovered in either the active or inactive form. However, even theproteolytic strains that produce, for example, the botulinum toxin typeB serotype only cleave a portion of the toxin produced. The exactproportion of nicked to unnicked molecules depends on the length ofincubation and the temperature of the culture. Therefore, a certainpercentage of any preparation of, for example, the botulinum toxin typeB toxin is likely to be inactive, possibly accounting for the knownsignificantly lower potency of botulinum toxin type B as compared tobotulinum toxin type A. The presence of inactive botulinum toxinmolecules in a clinical preparation will contribute to the overallprotein load of the preparation, which has been linked to increasedantigenicity, without contributing to its clinical efficacy.Additionally, it is known that botulinum toxin type B has, uponintramuscular injection, a shorter duration of activity and is also lesspotent than botulinum toxin type A at the same dose level.

High quality crystalline botulinum toxin type A can be produced from theHall A strain of Clostridium botulinum with characteristics of >3×10⁷U/mg, an A₂₆₀/A₂₇₈ of less than 0.60 and a distinct pattern of bandingon gel electrophoresis. The known Shantz process can be used to obtaincrystalline botulinum toxin type A, as set forth in Shantz, E. J., etal, Properties and use of Botulinum toxin and Other MicrobialNeurotoxins in Medicine, Microbiol Rev. 56;80-99:1992. Generally, thebotulinum toxin type A complex can be isolated and purified from ananaerobic fermentation by cultivating Clostridium botulinum type A in asuitable medium. The known process can also be used, upon separation outof the non-toxin proteins, to obtain pure botulinum toxins, such as forexample: purified botulinum toxin type A with an approximately 150 kDmolecular weight with a specific potency of 1-2×10⁸ LD₅₀ U/mg orgreater; purified botulinum toxin type B with an approximately 156 kDmolecular weight with a specific potency of 1-2×10⁸ LD₅₀ U/mg orgreater, and; purified botulinum toxin type F with an approximately 155kD molecular weight with a specific potency of 1-2×10⁷ LD₅₀ U/mg orgreater.

Botulinum toxins and/or botulinum toxin complexes can be obtained fromList Biological Laboratories, Inc., Campbell, Calif.; the Centre forApplied Microbiology and Research, Porton Down, U.K.; Wako (Osaka,Japan), Metabiologics (Madison, Wis.) as well as from Sigma Chemicals ofSt Louis, Mo. Pure botulinum toxin can also be used to prepare apharmaceutical composition.

As with enzymes generally, the biological activities of the botulinumtoxins (which are intracellular peptidases) is dependant, at least inpart, upon their three dimensional conformation. Thus, botulinum toxintype A is detoxified by heat, various chemicals surface stretching andsurface drying. Additionally, it is known that dilution of the toxincomplex obtained by the known culturing, fermentation and purificationto the much, much lower toxin concentrations used for pharmaceuticalcomposition formulation results in rapid detoxification of the toxinunless a suitable stabilizing agent is present. Dilution of the toxinfrom milligram quantities to a solution containing nanograms permilliliter presents significant difficulties because of the rapid lossof specific toxicity upon such great dilution. Since the toxin may beused months or years after the toxin containing pharmaceuticalcomposition is formulated, the toxin can stabilized with a stabilizingagent such as albumin and gelatin.

A commercially available botulinum toxin containing pharmaceuticalcomposition is sold under the trademark BOTOX® (available from Allergan,Inc., of Irvine, Calif.). BOTOX® consists of a purified botulinum toxintype A complex, albumin and sodium chloride packaged in sterile,vacuum-dried form. The botulinum toxin type A is made from a culture ofthe Hall strain of Clostridium botulinum grown in a medium containingN-Z amine and yeast extract. The botulinum toxin type A complex ispurified from the culture solution by a series of acid precipitations toa crystalline complex consisting of the active high molecular weighttoxin protein and an associated hemagglutinin protein. The crystallinecomplex is re-dissolved in a solution containing saline and albumin andsterile filtered (0.2 microns) prior to vacuum-drying. The vacuum-driedproduct is stored in a freezer at or below −5° C. BOTOX® can bereconstituted with sterile, non-preserved saline prior to intramuscularinjection. Each vial of BOTOX® contains about 100 units (U) ofClostridium botulinum toxin type A purified neurotoxin complex, 0.5milligrams of human serum albumin and 0.9 milligrams of sodium chloridein a sterile, vacuum-dried form without a preservative.

To reconstitute vacuum-dried BOTOX®, sterile normal saline without apreservative; (0.9% Sodium Chloride Injection) is used by drawing up theproper amount of diluent in the appropriate size syringe. Since BOTOX®may be denatured by bubbling or similar violent agitation, the diluentis gently injected into the vial. For sterility reasons BOTOX® ispreferably administered within four hours after the vial is removed fromthe freezer and reconstituted. During these four hours, reconstitutedBOTOX® can be stored in a refrigerator at about 2° C. to about 8° C.Reconstituted, refrigerated BOTOX® has been reported to retain itspotency for at least about two weeks. Neurology, 48:249-53:1997.

It has been reported that botulinum toxin type A has been used inclinical settings as follows: (1) about 75-125 units of BOTOX® perintramuscular injection (multiple muscles) to treat cervical dystonia;(2) 5-10 units of BOTOX® per intramuscular injection to treat glabellarlines (brow furrows) e.g., 5 units injected intramuscularly into theprocerus muscle and 10 units injected intramuscularly into eachcorrugator supercilii muscle); (3) about 30-80 units of BOTOX® to treatconstipation by intrasphincter injection of the puborectalis muscle; (4)about 1-5 units per muscle of intramuscularly injected BOTOX® to treatblepharospasm by injecting the lateral pre-tarsal orbicularis oculimuscle of the upper lid and the lateral pre-tarsal orbicularis oculi ofthe lower lid; (5) to treat strabismus, extraocular muscles have beeninjected intramuscularly with between about 1-5 units of BOTOX®, theamount injected varying based upon both the size of the muscle to beinjected and the extent of muscle paralysis desired (i.e. amount ofdiopter correction desired); (6) to treat upper limb spasticityfollowing stroke by intramuscular injections of BOTOX® into fivedifferent upper limb flexor muscles, as follows: (a) flexor digitorumprofindus: 7.5 U to 30 U; (b) flexor digitorum sublimus: 7.5 U to 30 U;(c) flexor carpi ulnaris: 10 U to 40 U; (d) flexor carpi radialis: 15 Uto 60 U; (e) biceps brachii: 50 U to 200 U. Each of the five indicatedmuscles has been injected at the same treatment session, so that thepatient receives from 90 U to 360 U of upper limb flexor muscle BOTOX®by intramuscular injection at each treatment session; (7) to treatmigraine, pericranial injected (injected symmetrically into glabellar,frontalis and temporalis muscles) injection of 25 U of BOTOX® has showedsignificant benefit as a prophylactic treatment of migraine compared tovehicle as measured by decreased measures of migraine frequency, maximalseverity, associated vomiting and acute medication use over the threemonth period following the 25 U injection. Any of the aforementioneddosages, along with dosages disclosed elsewhere in this application, areappropriate for use in the present invention.

Additionally, intramuscular botulinum toxin has been used in thetreatment of tremor in patient's with Parkinson's disease, although ithas been reported that results have not been impressive. Maijama-Jyons,J., et al., Tremor-Predominant Parkinson's Disease, Drugs & Aging16(4);273-278:2000.

It is known that botulinum toxin type A can have an efficacy for up to12 months (European J. Neurology 6 (Supp 4): S111-S1150:1999), and insome circumstances for as long as 27 months, when used to treat glands,such as in the treatment of hyperhydrosis. See e.g. Bushara K.,Botulinum toxin and rhinorrhea, Otolaryngol Head Neck Surg 1996;114(3):507, and The Laryngoscope 109:1344-1346:1999. However, the usualduration of an intramuscular injection of BOTOX® is typically about 3 to4 months.

The success of botulinum toxin type A to treat a variety of clinicalconditions has led to interest in other botulinum toxin serotypes. Twocommercially available botulinum type A preparations for use in humansare BOTOX® available from Allergan, Inc., of Irvine, Calif., andDysport® available from Beaufour Ipsen, Porton Down, England. ABotulinum toxin type B preparation (MyoBloc®) is available from ElanPharmaceuticals of San Francisco, Calif.

In addition to having pharmacologic actions at the peripheral location,botulinum toxins may also have inhibitory effects in the central nervoussystem. Work by Weigand et al, Nauny-Schmiedeberg's Arch. Pharmacol.1976; 292, 161-165, and Habermann, Nauny-Schmiedeberg's Arch. Pharmacol.1974; 281, 47-56 showed that botulinum toxin is able to ascend to thespinal area by retrograde transport. As such, a botulinum toxin injectedat a peripheral location, for example intramuscularly, may be retrogradetransported to the spinal cord.

A botulinum toxin has also been proposed for the treatment ofrhinorrhea, (chronic discharge from the nasal mucous membranes, i.e.runny nose), rhinitis (inflammation of the nasal mucous membranes),hyperhydrosis and other disorders mediated by the autonomic nervoussystem (U.S. Pat. No. 5,766,605), tension headache, (U.S. Pat. No.6,458,365), migraine headache (U.S. Pat. No. 5,714,468), post-operativepain and visceral pain (U.S. Pat. No. 6,464,986), pain treatment byintraspinal toxin administration (U.S. Pat. No. 6,113,915), Parkinson'sdisease and other diseases with a motor disorder component, byintracranial toxin administration (U.S. Pat. No. 6,306,403), hair growthand hair retention (U.S. Pat. No. 6,299,893), psoriasis and dermatitis(U.S. Pat. No. 5,670,484), injured muscles (U.S. Pat. No. 6,423,319,various cancers (U.S. Pat. No. 6,139,845), pancreatic disorders (U.S.Pat. No. 6,143,306), smooth muscle disorders (U.S. Pat. No. 5,437,291,including injection of a botulinum toxin into the upper and loweresophageal, pyloric and anal sphincters)), inflammation, arthritis andgout (U.S. Pat. No. 6,063,768), juvenile cerebral palsy (U.S. Pat. No.6,395,277), inner ear disorders (U.S. Pat. No. 6,265,379), thyroiddisorders (U.S. Pat. No. 6,358,513), parathyroid disorders (U.S. Pat.No. 6,328,977) and neurogenic inflammation (U.S. Pat. No. 6,063,768).Additionally, controlled release toxin implants are known (see e.g. U.S.Pat. Nos. 6,306,423 and 6,312,708).

Tetanus toxin, as wells as derivatives (i.e. with a non-native targetingmoiety), fragments, hybrids and chimeras thereof can also havetherapeutic utility. The tetanus toxin bears many similarities to thebotulinum toxins. Thus, both the tetanus toxin and the botulinum toxinsare polypeptides made by closely related species of Clostridium(Clostridium tetani and Clostridium botulinum, respectively).Additionally, both the tetanus toxin and the botulinum toxins aredichain proteins composed of a light chain (molecular weight about 50kD) covalently bound by a single disulfide bond to a heavy chain(molecular weight about 100 kD). Hence, the molecular weight of tetanustoxin and of each of the seven botulinum toxins (non-complexed) is about150 kD. Furthermore, for both the tetanus toxin and the botulinumtoxins, the light chain bears the domain which exhibits intracellularbiological (protease) activity, while the heavy chain comprises thereceptor binding (immunogenic) and cell membrane translocationaldomains.

Further, both the tetanus toxin and the botulinum toxins exhibit a high,specific affinity for ganglioside receptors on the surface ofpresynaptic cholinergic neurons. Receptor mediated endocytosis oftetanus toxin by peripheral cholinergic neurons results in retrogradeaxonal transport, blocking of the release of inhibitoryneurotransmitters from central synapses and a spastic paralysis.Contrarily, receptor mediated endocytosis of botulinum toxin byperipheral cholinergic neurons results in little if any retrogradetransport, inhibition of acetylcholine exocytosis from the intoxicatedperipheral motor neurons and a flaccid paralysis.

Without being bound by theory, the basis for the treatment of theneurological-urological conditions according to the instant invention isthe removal or modulation of the neural basis for the dysfunctionalregulation of the affected tissue. For example, the modulation of theneural basis of prostate glandular dysfunction can be accomplished byany non-surgical means known in the art. Such means can include, forexample, biofeedback, α-blockers, pharmacological methods, and the useof one or more neurotoxins to inhibit synaptic function in the affectedgland. It is preferred that the neurotoxin cause long-lasting inhibitionof synaptic function, preferably greater than one week, more preferablygreater than one month, most preferably six to eight months or longer.Such neurotoxins can include, for example, capsaicin, resinoferatoxin,a-bungotoxin, terodotoxin and botulinum toxin. Botulinum toxin is apreferred neurotoxin according to the instant invention, particularlybotulinum toxin A, more particularly BOTOX® (Allergen).

The toxin can be formulated in any pharmaceutically acceptableformulation in any pharmaceutically acceptable form. Such forms andformulations include liquids, powders, creams, emulsions, pills,troches, suppositories, suspensions, solutions, and the like. The toxincan also be used in any pharmaceutically acceptable form supplied by anymanufacturer.

In a preferred embodiment in accordance with the method of the instantinvention, the neurotoxin is botulinum toxin type A. Therapeuticallyeffective amounts of botulinum toxin can be any amounts or doses thatare less than a toxic dose, for example, less than about 3000 IU/70 kgmale or about 43 IU per kg, preferably between 100 IU/70 kg male orabout 1.4 IU per kg, up to about 1200 IU/70 kg or about 17 IU per kg.Preferred amounts of botulinum toxin to administer includes amountsbetween about 20 IU per 70 kg male or about 0.3 IU per kg, amounts ofabout 30 IU per 70 kg male or about 0.4 IU per kg, about 40 IU per 70 kgmale or about 0.6 IU per kg, to about 100 IU per 70 kg male or about 1.4IU per kg. The dosages can be given as a single dose, or as divideddoses, for example, divided over the course of four weeks.

In another embodiment, if the neurotoxin is botulinum toxin type B, thedosage is approximately 50 times greater than the functionallyequivalent dosage of botulinum toxin type A. For example, dosages ofbotulinum toxin type B can include dosages of at least about 20 I.U. perdose to about 1000 I.U. per dose, and so in, increments of 10 I.U.(i.e., 30 I.U., 40 I.U., . . . 100 IU, 110 IU.), and so on, up to 10,000I.U. or greater.

The neurotoxins of the instant invention can be administered by anysuitable means. In the preferred embodiment of the invention, botulinumtoxin is administered by injection. Such injection can be administeredto any affected area. For example, in one embodiment, the neurotoxin canbe injected urethroscopically into the prostate with 200 IU with singleor serial dosing. For example, one suggested course of treatment for BPHand other conditions that can be treated using the method of theinvention is 200 units of BOTOX® every 3-4 months, as indicated bytherapeutic requirements. Preferably the neurotoxin is injected everythree days until a therapeutic effect is achieved or up to about 2500units.

The following techniques are used in this invention:

Tissue Preparation for Light Microscopy

Tissues are fixed in 6% paraformaldehyde in 0.1 M phosphate buffer, pH7.2, for 24 hours, dehydrated in graded alcohol and xylene, and embeddedin paraffin. Sections are cut and stained with appropriate stains, suchas hematoxylin/eosin.

Tissue Preparation for Election Microscopv

Tissues are collected and fixed in 2.5% glutaraldehyde in 0.1 Mphosphate buffer, pH 7.2, for 1 hour at 4° C., then incubated with 0.1%osmium tetroxide for 1 hour and embedded in EPON. Ultrathin sections (80nm) are prepared and stained with lead citrate/uranyl acetate andexamined vrith an electron microscope (philips, model 201).

Tunel Stain for Apoptosis

The tissue is fixed and embedded as described above. The tissues aredeparaffinized and reacted with Proteinase K (Boehringer). They arefurther treated with peroxidase and TDT enzyme and placed in ahumidifier set at 30° C. for one hour. The sections are washed andanti-digoxigenin-peroxidase is added for 30 minutes, followed bystaining with nickel-DAB (diaminobenzene).

Immunohistochemistry Studies

The presence of the neuropeptides VIP, SP, NYP, L-Enk and calcitoningene-related peptide (CGRP) as well as the expression of transforminggrowth factor beta (TGF-beta), transforming growth factor alpha(TGF-alpha), epidermal growth factor (EGF) and basic fibroblast growthfactor (bFGF) are determined in prostatic tissues using appropriatemonoclonal antibodies. Use of neurotoxins results in prostatic atrophy,which should be reflected by lower levels of growth factors in treatedprostatic tissue.

Sections are incubated overnight at room temperature with primaryantibodies followed by immunostaining with avidin-biotin-peroxidase(Vectastain Elite ABC, Vector Labs, USA). Rabbit polyclonal antiserumagainst the neurotransmitters VIP, CGRP, SP,NPY and L-Enk (PeninsulaLabs, USA) is used in these preparations, at dilutions of 1:8000 to1:12,000. Immunocytochemical controls consist of preabsorbing theprimary antiserum with appropriate antigen, or their substitution withnormal serum (Blasi, J., Chapman, E. R., Yamas, S., Binz, T., Niemann, Hand Jahn, R. [1993] The EMBO Journal 12: 4821-4828; Black, J. D. andDolly, J. O. [1986] J. Cell Biol. 103; 535-544; Linial, M. [1995] Is. J.Med. Sci. 31: 591-595). After mounting on slides, sections arecounterstained with eosin, dehydrated and coverslipped.

Western Blot Analysis of Growth Factor Expression

Treated and untreated prostate cell homogenates are examined forexpression of growth factors by Western blot analysis. Cell homogenateprotein is separated by electrophoresis on SDS-PAGE (7%), thentransferred electrophoretically overnight to nitrocellulose paper(Towbin, H., et al., [1979] Proc. Nat. Acad. Sci. 76(9): 4350-4379). Thenitrocellulose paper is soaked for one hour at room temperature in 0.5%non-fat dry milk dissolved in phosphate buffered saline, and furthersoaked overnight at 4° C. in blocking solution (2% bovine serum albuminin 10 mM Tris/0.15 M NaCl/0.1% sodium azide, pH 7.4). The nitrocellulosemembranes are incubated with antibodies (IgG fractions of anti-TGF-beta,anti-TGF-alpha, anti-EGF and anti-bFGF) purified by protein A (1×10⁶cpm/ml) in blocking buffer for 1 hour. The membrane is washed with PBScontaining Nonidet P-40 between incubations. X-O-mat AR2 film (Kodak) isexposed to the membrane at −70° C. and films are developed to examinethe expression of growth factors.

Determination of c-fos and c-myc Expression

Expression of c-fos and c-myc in treated and untreated prostatic tissueis determined by Northern blot analysis as follows. Tissue ishomogenized in lysis buffer for 15 seconds or until the tissuehomogenizes. Sodium acetate is added and the solution is mixed byswirling. An equal volume of water-saturated phenol is added and mixedby inversion, followed by addition of chloroform/isoamyl alcohol. Thesolution is vortexed vigorously for 30 seconds, and allowed to settle onice for 15 minutes. The solution is centrifuged for 10-20 minutes at 4°C. After centrifugation, the aqueous phase is carefully aspirated andplaced in a new polypropylene tube. One volume of isopropanol is addedand the solution is mixed by swirling. The solution is placed in a −20°C. freezer for at least 60 minutes to precipitate RNA. Afterprecipitation, the tube is centrifuged for 10 minutes, and thesupernatant is decanted, leaving the RNA pellet. One ml of ethanol isadded, and the tube is centrifuged for additional 10 minutes. Theaqueous phase is discarded, and the pellet is washed with 100% ethanolby vortexing. The RNA pellet is redissolved in 0.4 ml of lysis buffer.The RNA is re-precipitated by the addition of 100% ethanol andincubation at −20° C. freezer for at least 60 minutes. The solution iscentrifuged and the supernatant discarded. RNA concentration isdetermined by diluting 5 μL of sample into 995 μL of DEPC water andmeasuring the ratio of absorbance at 260/280 nm.

The following examples are provided by way of describing specificembodiments without intending to limit the scope of the invention in anyway.

EXAMPLE 1

This Example describes denervation of the prostate.

Unilateral denervation of the prostate is carried out by removal of thepelvic ganglia, which overlie the prostate of the rat. This approachpreserves the functional integrity of the bladder and posterior urethraand removes the possibility for artifact arising from major disturbancesin blood flow or micturation. Control animals undergo sham operationswithout concurrent denervation of the prostate. After denervation, theanimals are allowed to recover and maintained prior to collection of theprostate. The prostate is preserved, prepared for light microscopy andexamined histologically. The major findings are (1) reduced epithelialcell height primarily due to a decrease in the clear supranuclear zone(due to a reduction in the amount and size of the apical cistemae andthe endoplasmic reticulum); (2) major changes in protein expression onSDS gel electrophoresis (the endoplasmic reticulum is important inprotein synthesis) (3) modest reduction in the number of secretorygranules; (4) an increase in intracellular vacuoles, intercellular emptyspaces and reduction in microvilli on the cell surface; and (5) asignificant increase in nerve growth factor (NGF) content ipsilateral tothe denervation relative to the control group (188±10 vs. 46±20 vs.29±16 pg/g wet tissue (±SD) NGF is known to influence only sympatheticand sensory neurons. N=15 in both the control and experimental groups.

EXAMPLE 2

This Example describes the effect of neurotoxin injection on normalprostate: rat prostate.

Rats were randomly assigned into three groups. The first group receiveda single acute dose of Botulinum toxin type A (BOTOX® Allergen) of 5, 10or 15 IU. These animals were sacrificed one week after injection. Thesecond group received a series of 4 weekly injections of 5 IU ofBotulinum toxin and were sacrificed at 5 weeks. Control rats receivedsaline injections. Injections were performed as single or serialinjections into the left and/or right ventral lobe of the prostate. Notethat an injection of methylene blue into one lobe of the rat prostateshowed immediate diffusion into the opposite lobe. Thus, there wascommunication between the prostate lobes and therefore the contralaterallobe could not be used as a true comparative control.

The weight of each prostate ventral lobe collected from healthy animalswas approximately 0.50 gram. All toxin-treated animals showed shrinkageof prostate volume, first in the injected lobe, and with subsequentinjections, reduction in the overall volume. After four serialinjections, the left prostate lobe weighed 0.12-0.17 gram, while theright lobe weighed 9.10-0.14 grams. This represented a reduction of overtwo-thirds of the original size.

EXAMPLE 3

This Example describes the effect of neurotoxin injection on urologicaldysfunctions: human data.

Three patients with recalcitrant voiding dysfunction were treated withinjections of botulinum toxin (BOTOX®) as follows. Patient 1 was a47-year-old male who was incontinent secondary to an injury sustained atthe cervical vertebrae (level C6-C7) sustained 14 months previously.Urodynamics on presentation revealed a bladder capacity of 30 cc and aweak sphincter (peak urethral pressure of 40 cm water). He had failedmultiple pharmacological regimes and was intolerant to penileclamp/condom devices.

He received four weekly 200 IU botulinum toxin injections into thebladder neck for total dose of 800 IU. Post-injection, his bladdercapacities ranged from 300-400 cc with oxybutinin and 150-200 cc withoutOxybutinin. Peak bladder pressures pre-injection had been 200-cm water,compared to post injection bladder pressures of 40 cm of water. Thepatient was continent with a penile clamp after treatment with botulinumtoxin. In addition, walking and erections improved due to reducedbladder spasticity.

Patient 2 was a 55 year old T12 paraparetic female secondary totraumatic injury 14 years previous. The patient presented with urgeincontinence, and had been on self-catheterization every 2 hours duringthe day and two times at night. The patient received injections into thelateral bladder wall in two weekly injections of 200 IU each for a totalof 400 IU of botulinum toxin. The patient's voiding diary data revealedpre-injection capacities of between 150-200 cc. Post injection, diarydata indicated bladder capacity increased to 300-400 cc. In addition,the patient no longer had annoying constant urge type dysfunction, sleptthrough the night and was continent on self-catheterization every 4hours.

Patient 3 was a 65 year old male with disabling perineal pain followingradiation treatment for prostatic cancer. The patient had failed medicaltherapy. He was treated with one 200 IU injection of botulinum toxininto the external urethral sphincter. The patient experienced dramaticrelief of testicle pain and had far less severe pain in the shaft of thepenis. Erections were not affected.

EXAMPLE 4

This Example describes the determination of the smallest effective dose.

Rats are injected in the prostate ventral lobes with single and serialdoses of botulinum toxin (BOTOX®). The prostates are harvested atdifferent time intervals to determine the smallest effective dose, aswell as the morphological and physiological changes taking place withtime. The smallest effective dose is defined as that dose that woulddemonstrate a decrease in prostate volume.

To assess the response to electrical field stimulation, preparations aremounted between two platinum electrodes placed in the organ bath. Thetension of the preparations is adjusted. Transmural stimulation ofnerves is performed using a Danted Neuromatic 2000 Simulator deliveringsingle-wave pulses at supramaximal voltage with a duration of 0.8milliseconds at a frequency of 0.5 to 80 hertz. The polarity of theelectrodes is changed after each pulse by means of a polarity-changingunit. The train duration is five seconds and the train interval 120seconds. Isometric tension is recorded by using a Gould thermo-array8-channel recorder. Separate experiments are performed to determine thepreload teflsion producing optimal responses. In addition, the effect ofthe electric field stimulation in the presence of differentconcentrations of individual neuropeptides is determined. Theseneuropeptides are 10-20 μM adrenaline, 10 μM clonidine, 5-50 mMregitine, 10 nM-0.1 μM acetylcholine, 1-3 μM atropine, 1 nM-10 μMnifedipine, 1-10 nM VIP and 1-250 nM NPY. The effect of nitroprusside (anitric oxide releasing substance) and methylene blue (a guanylatecyclase inhibitor) on prostate tone and contraction resulting from fieldstimulation also is examined in these tissues.

EXAMPLE 5

This Example describes the effect of botulinum toxin on rat prostatictissue: comparison of hormonally intact rats to hormonally deprivedrats.

To determine if there is any interaction between the neurotoxin andtesticularly-derived hormones, studies are performed which will examinethe interaction of the neurotoxin with hormonal components. Thesestudies will compare prostatic tissue treated with botulinum toxinharvested from rats that have undergone orchiectomy (hormonally depletedrats) and prostatic tissue from rats treated with botulinum toxin thatdid not undergo orchiectomy. Fifty-two age-matched rats are treated asdescribed below. Four healthy rats will undergo a sham operationconsisting of anesthesia induction, exposure of the prostate andinjection of 0.2 cc saline into the left ventral lobe of the prostate.Three rats are given bilateral orchiectomy with no injection to theprostate (hormonally depleted controls), five rats will have orchiectomyand injection of 0.2 ml saline in the left ventral lobe (hormonaldepletion+surgical stress control). Four groups of rats receivebotulinum injections of 0.5 IU, 1.0 IU, 1.5 IU and 2.5 IU only(hormonally intact experimental rats). Sixteen rats undergo bilateralorchiectomy. Eight of these rats are treated with a single injection of2.5 IU botulinum toxin into the left ventral lobe 5 weeks after surgery.All rats are sacrificed after six weeks, and the harvested prostate isprepared for examination as described above. A similar atrophic effecton glandular epithelium is expected.

EXAMPLE 6

This Example describes the effects of botulinum toxin on patients.

Patients affected by benign prostatic hyperplasia, abacterial prostatis,or prostatodynia are studied both before and after treatment withbotulinum toxin. Patients are eligible for inclusion in this study ifthey are affected by BPH between the ages of 40 and 80, or if they arebetween 25 and 60 and have been diagnosed with abacterial prostatitis orprostatodynia. Preferred patients are those who are not good surgicalcandidates. Patients are evaluated prior to treatment by determinationof prostate specific antigen levels (PSA), evaluation of urodynamicparameters (cystometrogram, urethral pressure profile and flowmetry),determination of American Urological Association (AUA) symptom score(Barry, M. J., et al., [1992] J. Urol, 148: 1549-1557), maintenance of avoiding diary, and examination of the prostate by transrectal ultrasoundwith biopsy (for BPH patients only). One week after initial evaluationis completed, the patient is injected urethroscopically with 200 IU ofbotulinum toxin as either single unilateral injections, serialunilateral injections or 1.5 bilateral injections. BPH patients aretreated by TURP or undergo control TURP-biopsy 7 days after singleinjection or 5 weeks after serial injections. The harvested prostatictissues are prepared for examination as described in the previousExamples. The patients are re-evaluated after injection using the sameparameters examined during the initial evaluation.

EXAMPLE 7

This example describes treatment of patients with hyperreflexive bladderdue to neurogenic bladder dysfunction.

Several patients with hyperreflexive bladders with symptoms includingbladder infection, incontinence, and urge incontinence due to neurogenicbladder dysfunction as a result of spinal cord injury will be treated byinjection of an effective amount of BOTOX®, for example, preferablyabout 20-30 IU or up to 100-200 IU into the detrusor muscle of thebladder. The BOTOX® is divided up into multiple doses, such as, forexample, 10 doses, and injected into multiple sites on the bladder,sparing the trigone. Patients will be under light sedation. Asignificant increase in the mean maximum bladder capacity andsignificant decrease in the mean maximum detrusor voiding pressure isexpected post-injection. It is predicted that maximal efficacy ofbotulinum injection is achieved within seven days post injection. It isexpected that most patients will report that both sleep quantity andquality improved, and also will report a decrease or absence ofincontinence and a significant decrease in voiding symptoms. Additionaldosing with BOTOX® may be performed if required for maximal response.Clinical responses are predicted to last from four to more than one year(e.g., fourteen months) with no adverse effects with the treatment beingobserved.

EXAMPLE 8

This Example describes treatment of patients with difficulty relaxingthe urinary sphincter muscle.

A patient with multiple sclerosis is diagnosed with neurogenic bladderdysfunction after presenting with urge-type dysfunction, and/ordifficulty in emptying the bladder. The patient may also suffer fromrepeated urinary tract infections due to retained urine in the bladder.The patient is treated with an effective amount of BOTOX®, for example,preferably about 20-30 IU or up to 100-200 IU of BOTOX® into theexternal sphincter. Treatment will consist of multiple transurethralinjections (such as, for example, four injections of 2.5 milliliterseach) spaced equally around the sphincter at the level of the striatedsphincter. Injections will be directed deeper than collagen injectionsto target nerve terminals innervating skeletal muscle. It is expectedthat within a matter of days, the patient's bladder functioning will benormal or near-normal and the patient will report a disappearance and/orreduction of symptoms. Additional dosing with BOTOX® may be performed ifrequired for maximal response. It is expected that the patient willreport that symptoms do not begin to reappear until six months or morehave passed. It is then expected that treatment described above can berepeated successfully.

The foregoing description of the invention is exemplary for purposes ofillustration and explanation. It will be apparent to those skilled inthe art that changes and modifications are possible without departingfrom the spirit and scope of the invention. All documents cited hereinare hereby incorporated by reference. It is intended that the followingclaims be interpreted to embrace all such changes and modifications.

1. A method for treating a patient with a neurogenic bladderdysfunction, said method comprising the step of injecting atherapeutically effective amount of botulinum toxin type A into thelateral bladder wall of a patient with said neurogenic bladderdysfunction, thereby treating a symptom of said neurogenic bladderdysfunction.
 2. The method of claim 1, wherein said neurogenic bladderdysfunction is secondary to a disease condition selected from the groupconsisting of Parkinson's disease, a spinal cord injury, a stroke,multiple sclerosis, and a spasm reflex.
 3. The method of claim 1,wherein the step of injection comprises a single injection.
 4. Themethod of claim 1, wherein the step of injection comprises injecting agel comprising the botulinum toxin.
 5. The method of claim 1, whereinthe step of injecting results in increased bladder capacity.
 6. Themethod of claim 1, wherein the therapeutically effective amount ofbotulinum toxin type A is up to 2500 units.
 7. The method of claim 1,wherein the therapeutically effective amount of botulinum toxin type Ais about 1.4 IU/kg to 17.1 IU/kg of botulinum toxin type A.
 8. Themethod of claim 1, wherein the therapeutically effective amount ofbotulinum toxin type A is 200 IU of botulinum toxin type A.